It is our long-range goal to clarify the nature of the cellular molecules responsible for the malignant phenotype of chemically transformed human cells by using new techniques in molecular biology. The mutated beta-action, which we had found previously in a chemically transformed human fibroblast cell line, was further altered in subclones of the transformed cells. Variations in the mutated beta-actin expression were accompanied by incremental increases of the malignant potential in the subclones. The expression of this mutated beta-actin was also associated with the expression of in vitro transformed phenotypes in hybrid cells between transformed and normal parent human fibroblasts, suggesting an important role for the mutated beta-actin in the expression of transformed phenotypes. Computerized microdensitometry of autoradiographs of two-dimensional gel electrophoresis of the polypeptide revealed that less than 2% of the genes were activated or shut off, but at least 32% were modulated quantitatively as a consequence of neoplastic transformation of human fibroblasts by a chemical carcinogen. A human cardiac muscle actin gene was isolated from a human DNA library and its molecular structure and evolutionary origin was determined.